Receptor for Activated C Kinase1B (OsRACK1B) Impairs Fertility in Rice through NADPH-Dependent H2O2 Signaling Pathway

The scaffold protein receptor for Activated C Kinase1 (RACK1) regulates multiple aspects of plants, including seed germination, growth, environmental stress responses, and flowering. Recent studies have revealed that RACK1 is associated with NADPH-dependent reactive oxygen species (ROS) signaling in plants. ROS, as a double-edged sword, can modulate several developmental pathways in plants. Thus, the resulting physiological consequences of perturbing the RACK1 expression-induced ROS balance remain to be explored. Herein, we combined molecular, pharmacological, and ultrastructure analysis approaches to investigate the hypothesized connection using T-DNA-mediated activation-tagged RACK1B overexpressed (OX) transgenic rice plants. In this study, we find that OsRACK1B-OX plants display reduced pollen viability, defective anther dehiscence, and abnormal spikelet morphology, leading to partial spikelet sterility. Microscopic observation of the mature pollen grains from the OX plants revealed abnormalities in the exine and intine structures and decreased starch granules in the pollen, resulting in a reduced number of grains per locule from the OX rice plants as compared to that of the wild-type (WT). Histochemical staining revealed a global increase in hydrogen peroxide (H2O2) in the leaves and roots of the transgenic lines overexpressing OsRACK1B compared to that of the WT. However, the elevated H2O2 in tissues from the OX plants can be reversed by pre-treatment with diphenylidonium (DPI), an NADPH oxidase inhibitor, indicating that the source of H2O2 could be, in part, NADPH oxidase. Expression analysis showed a differential expression of the NADPH/respiratory burst oxidase homolog D (RbohD) and antioxidant enzyme-related genes, suggesting a homeostatic mechanism of H2O2 production and antioxidant enzyme activity. BiFC analysis demonstrated that OsRACK1B interacts with the N-terminal region of RbohD in vivo. Taken together, these data indicate that elevated OsRACK1B accumulates a threshold level of ROS, in this case H2O2, which negatively regulates pollen development and fertility. In conclusion, we hypothesized that an optimal expression of RACK1 is critical for fertility in rice plants.

Introduction
The receptor for Activated C Kinase1 (RACK1) is a WD-40 type transmembrane protein that is ubiquitously expressed in different tissues and organs in plants and participates in several biological processes ranging from seed germination, root formation, growth, leaf vegetation, flowering, and fruit ripening to senescence [1][2][3][4]. As a scaffold protein, RACK1 regulates several signaling pathways through interactions with hundreds of proteins in eukaryotic systems [5]. Although it is less clear how RACK1 contributes to diverse biological processes, a dynamic and inducible modification at both the transcriptional and the translational levels has been proposed as a key to RACK1 s functionality [6][7][8].
Early genetic studies demonstrated RACK1 s role in environmental stress signaling and hormone responses. Current evidence focuses on a new insight into RACK1 s role in plant immune signaling and has identified RACK1 as a regulator of reactive oxygen species (ROS)-mediated systemic signaling. For example, rice RACK1 (OsRACK1) is shown to be involved in the immune response against pathogen attack through enhanced reactive oxygen species (ROS), such as H 2 O 2 production regulated by the Rac1 immune complex [9]. OsRACK1 forms a complex with the GTP form of Rac1 and triggers NADPH oxidasedependent ROS bursts (in plants, NADPH oxidase (NOX) is also known as respiratory burst oxidase homolog, Rboh) in response to blast fungus. At the same time, Li et al. showed that RACK1 negatively regulates the redox system-related tolerance to drought stress in transgenic rice plants, and superoxide dismutase activity (SOD) is significantly higher in rack1a mutant plants than in normal plants [10]. Zhang et al. have shown that OsRACK1 positively regulates rice seed germination by controlling endogenous hydrogen peroxide (H 2 O 2 ), and several Rboh transcripts were differentially expressed in transgenic rice plants [4].
RBOHs encode NADPH oxidases that localize at the plasma membrane to produce O 2 •-, which then mostly converts to apoplastic H 2 O 2 [11][12][13]. Studies have demonstrated that the spatiotemporal expression of RBOH proteins inducing ROS bursts is highly regulated by multiple factors, such as Ca 2+ influx, phosphorylation by calcium-dependent protein kinases (CPKs) and mitogen-activated protein kinase (MAPK) elements, transcriptional reprogramming, and phytohormone regulation [14,15]. The evidence also suggests that cytosolic calcium concentration plays an important role in modulating the NADPH oxidase activity by binding the EF-hand motifs of the N-terminal region of RBOH [16]. A tight balance between the dynamic regulation of RBOH proteins and the ROS bursts is shown to be essential for plant growth and development, specifically for pollen development, pollen tube elongation, and cell wall integrity [17][18][19][20][21][22][23].
Three separate studies in maize, soybean, and kale suggest that the overexpression of RACK1 induces Rboh-dependent H 2 O 2 production to regulate pathogen, drought, and salt resistance, respectively [39][40][41]. These reports uncovered shared signaling components and a direct crosstalk between RACK1 and H 2 O 2 signaling cascades in response to environmental cues. Yet, the dynamics and physiological outcomes of such complex interactions remain elusive. The rice genome contains two RACK1 homologous genes: OsRACK1A and OsRACK1B with 82% amino acid sequence homology [9].
In this study, we used the rice T-DNA insertion activation-tagging mutagenesis approach to characterize the function of OsRACK1B. We explored a possible mechanism of how RACK1 cross-talks with ROS signaling components and its potential physiological outcome. We found that the overexpression of OsRACK1B induces an Rboh-mediated ROS burst as a form of H 2 O 2 through direct interaction with the N-terminal region of RbohD. Our work indicates that the apoplastic systemic ROS produced by NADPH oxidases, together with the controlled antioxidant enzyme activity, is critical to ensuring the proper timing of anther dehiscence, pollen viability, and pollen cell wall integrity. We hypothesize that an optimal level of OsRACK1B is essential for pollen development and that OsRACK1B helps to maintain pollen cell wall integrity by maintaining a threshold level of H 2 O 2 .

Identification of T-DNA Insertion Activation-Tagged Rice Plants Overexpressing OsRACK1B
For the functional characterization of OsRACK1B (Loc_Os05g47890), we screened overexpression lines from the RICEGE database (http://signal.salk.edu/cgi-bin/RiceGE, accessed on 4 April 2012). We identified the PFG_3A-60871.L, PFG_3A-07870.R lines as the putative gain-of-function lines where a quadruple CaMV 35S enhancer is located at 3 -UTR of Loc_Os05g47890 on chromosome 5 in opposite directions. PCR genotyping analysis confirmed the presence of T-DNA in several seedlings. Expression analysis by qRT-PCR and Western blot revealed that two plants from putative gain-of-function lines accumulated 5-7-fold more OsRACK1B transcripts and higher protein levels than those in Dongjin (hereafter termed WT) plants ( Figure 1B,D). We refer to these two lines as the OsRACK1B overexpressed lines and designated them OX-1 (PFG_3A-07870.R) and OX-2 (PFG_3A-60871.L). Sequence analysis indicated that in both plants the T-DNA insertion was located in the 3 UTR region after the stop codon (TGA) at the 3 end ( Figure 1A, schematic diagram). Within the nearly 10 kb insertion site, except for several transposons, no other gene coding sequences could be seen, implying a low possibility of interference by the ectopic activation of other nearby genes.

Identification of T-DNA Insertion Activation-Tagged Rice Plants Overexpressing OsRACK1B
For the functional characterization of OsRACK1B (Loc_Os05g47890), we screened overexpression lines from the RICEGE database (http://signal.salk.edu/cgi-bin/RiceGE, accessed on 4 April 2012). We identified the PFG_3A-60871.L, PFG_3A-07870.R lines as the putative gain-of-function lines where a quadruple CaMV 35S enhancer is located at 3'-UTR of Loc_Os05g47890 on chromosome 5 in opposite directions. PCR genotyping analysis confirmed the presence of T-DNA in several seedlings. Expression analysis by qRT-PCR and Western blot revealed that two plants from putative gain-of-function lines accumulated 5-7-fold more OsRACK1B transcripts and higher protein levels than those in Dongjin (hereafter termed WT) plants ( Figure 1B,D). We refer to these two lines as the OsRACK1B overexpressed lines and designated them OX-1 (PFG_3A-07870.R) and OX-2 (PFG_3A-60871.L). Sequence analysis indicated that in both plants the T-DNA insertion was located in the 3′UTR region after the stop codon (TGA) at the 3′end ( Figure 1A, schematic diagram). Within the nearly 10 kb insertion site, except for several transposons, no other gene coding sequences could be seen, implying a low possibility of interference by the ectopic activation of other nearby genes.  Samples were collected from four-week-old wild-type (WT) and OsRack1b mutant rice plants. For Western blot, an equal amount of leaf proteins extracted from the indicated genotypes was subjected to immunoblot analysis stained with anti-OsRACK1B antibody. Ponceau-stained membrane is shown as the loading control. Molecular weight markers are indicated in kDa. For qRT-PCR analysis, the normalized expression level of OsRACK1B in transgenic rice lines was compared to the wild-type plant. Total RNA was extracted from leaf tissues sampled from detached leaves of four-week-old wild-type and transgenic plants, as described in Methods. OsActin-1(LOC4333919) was used to standardize transcript levels in each sample. The data are shown as the means ± SE of three technical repeats. Double, and triple asterisks indicate statistical significance: ** p < 0.01 and *** p < 0.001, respectively, compared to wild-type (Student's t-test). Primers are listed in Table S2.

OsRACK1B Modulates RBOH-Dependent H 2 O 2 Accumulation
Compelling evidence has indicated that the overexpression of RACK1 in rice (Os-RACK1A), maize (ZmRACK1), kale (BoRACK1), and soybean (GmRACK1) plants leads to elevated ROS production [39][40][41]. Therefore, we investigated the oxidation states of the transgenic rice plants using 3,3 -diaminobenzidine (DAB) and 2 ,7 dichlorodihydrofluorescein diacetate acetyl ester (H 2 DCFDA) staining methods to determine the accumulation of H 2 O 2 in transgenic and wild-type plants. We found that leaves from OsRACK1Boverexpressed plants accumulate more H 2 O 2 , visible as dark brown spots due to H 2 O 2specific DAB polymer precipitation [42], than that of the wild-type plants ( Figure 2C). To investigate the source of the intracellular accumulation of H 2 O 2 , we stained leaves with H 2 DCFDA alone and with diphenyleneiodonium (DPI), which specifically inhibits superoxide radical generation by RBOH proteins [27,43]. Consistent with the DAB staining, microscopic analysis of the H 2 DCFDA staining (green fluorescence) revealed significant H 2 O 2 accumulation inside the cells of RACK1B-OX rice leaf sheaths and roots compared to that of the wild-type ( Figure 1A,B). However, OsRACK1B-OX leaf sheaths and roots that were pretreated with DPI showed a distinct inhibition of H 2 O 2 accumulation. These findings suggested that RACK1B activates the Rboh-dependent H 2 O 2 production.

OsRACK1B Regulates Rboh and SOD Activity
Given that H 2 O 2 accumulation was observed in OsRACK1B-OX plants, we investigated the in-gel activity assay of crude protein extracts from the leaves of transgenic and WT plants. Interestingly, the gel activity using NAPDH as a substrate revealed a different migration pattern in the OsRACK1B-OX samples than that of the wild-type ( Figure 3A). While the WT fractionated samples produced a formazan band of 80 to 75 kD molecular masses, the OsRACK1B-OX samples had approximately 103 kD-sized bands. As the RBOH activity assay is based on the migration of enzyme complexes through native gels, we conclude that the RBOH enzyme activity in OsRACK1B-OX leaves is different to that of the wild-type plant. To further confirm the Rboh assay, the WT sample was tested for sensitivity to DPI (Rboh/NADPH oxidase inhibitor, 50 µM), and we observed a complete inhibition of Rboh activity ( Figure 3A, lane 4, +DPI). These data provide further evidence that OsRACK1B is regulating OsRboh activity, possibly at the post-transcriptional or post-translational levels.
Remarkably, several reports suggested that overexpression of Rboh does not result in constitutive ROS production but, rather, a post-transcriptional or post-translational processing such as phosphorylation that activates Rboh for the ROS burst [13,37,[44][45][46]. We further examined the transcription levels of two previously reported rboh genes: RbohB and RbohD, which are responsible for ROS production in rice. The qRT-PCR results revealed that the RbohD transcript levels were significantly increased in RACK1B-OX plants ( Figure 3D), while the RbohB expression (Supplemental Figure S3) was lower than that of the WT plant. We further confirmed the increased expression of OsRBOHD protein in both RACK1B-OX plants compared to WT, using Anti-RBOHD antibody ( Figure 3B).  [13,37,[44][45][46]. We further examined the transcription levels of two previously reported rboh genes: RbohB and RbohD, which are responsible for ROS production in rice. The qRT-PCR results revealed that the RbohD transcript levels were significantly increased in RACK1B-OX plants ( Figure 3D), while the RbohB expression (Supplemental Figure S3) was lower than that of the WT plant. We further confirmed the increased expression of OsRBOHD protein in both RACK1B-OX plants compared to WT, using Anti-RBOHD antibody ( Figure 3B). Rboh activation is attributed to the reactive O2 •-that is immediately dismutated by superoxide dismutase (SOD) enzymes to form H2O2 in the cytoplasm, mitochondria, and chloroplasts [47,48]. Therefore, we examined two important scavenging enzymes: MnSOD and CuZn-SOD activity. Manganese containing SOD (MnSOD) is localized in the mitochondria; copper and zinc-containing SOD (CuZnSOD) is located in the cytoplasm and the nucleus [49,50]. In-gel analysis revealed that the CuZn-SOD and MnSOD activities in the transgenic samples were more elevated than those of the WT plant ( Figure 3C). Catalase possesses a high affinity for H2O2 and catalyzes its dismutation into H2O and O2. Our qRT-PCR analysis showed that Catalase B transcripts were induced in transgenic plants compared to those of the WT plant ( Figure 3E). Combinedly, these data corroborate our observation of excessive H2O2, which is partly due to the increased activity of the antioxidant enzyme activities.  Rboh activity. Rice leaves from OsRACK1B overexpressed and WT plants were fractionated by denaturing PAGE and assayed for Rboh activity. Fifty micromolar of Rboh inhibitor, DPI, was added to the microsomal fraction of leaf from wild-type rice plant before loading in the designated well of the native PAGE gel indicated in the Rboh activity gel. The straight and dotted lines in RBOH activity gel indicate the different migration patterns of the Rboh enzyme. (B) Western blot analysis of OsRBOHD protein showing higher accumulation of the protein compared to the WT. An equal amount of leaf proteins extracted from the indicated genotypes was subjected to immunoblot analysis stained with anti-OsRBOHD antibody. Ponceau-stained membrane is shown as the loading control. Molecular weight markers are indicated in kDa. (C) The activity of SOD isozymes; MnSOD and CuZnSOD from total proteins extracted from OX and WT plants. SODs were separated by native PAGE on 12% (w/v) polyacrylamide gels, and gels were stained by the photochemical nitroblue tetrazolium method and scanned using flat-bed scanner. (D,E) qRT-PCR analysis shows elevated transcript levels of OsRbohD and OsCatB genes. The transcript levels were normalized using OsActin-1 (LOC4333919). The data are shown as the means ± SE of three technical repeats. Single, double, and triple asterisks indicate statistical significance: * p < 0.05, ** p < 0.01, and *** p < 0.001, respectively, compared to wild-type (Student's t-test). Primers are listed in Table S2.

Rboh activation is attributed to the reactive O 2
•that is immediately dismutated by superoxide dismutase (SOD) enzymes to form H 2 O 2 in the cytoplasm, mitochondria, and chloroplasts [47,48]. Therefore, we examined two important scavenging enzymes: MnSOD and CuZn-SOD activity. Manganese containing SOD (MnSOD) is localized in the mitochondria; copper and zinc-containing SOD (CuZnSOD) is located in the cytoplasm and the nucleus [49,50]. In-gel analysis revealed that the CuZn-SOD and MnSOD activities in the transgenic samples were more elevated than those of the WT plant ( Figure 3C). Catalase possesses a high affinity for H 2 O 2 and catalyzes its dismutation into H 2 O and O 2 . Our qRT-PCR analysis showed that Catalase B transcripts were induced in transgenic plants compared to those of the WT plant ( Figure 3E). Combinedly, these data corroborate our observation of excessive H 2 O 2 , which is partly due to the increased activity of the antioxidant enzyme activities.

OsRACK1B Interacts with N-terminus RbohD
Next, we investigated whether OsRboh is regulated by OsRACK1B through direct interaction in plants using bimolecular fluorescence complementation (BiFC) assays. Crystal structure and mutational analysis revealed that Rboh protein activity is regulated through its conserved cytoplasmic N-terminal region containing two EF-hand motifs [51]. In vitro binding and NMR assay revealed that the N-terminal region of RBOH forms intramolecular interactions in the C-terminal region to prevent homodimerization [51][52][53]. Indeed, using split ubiquitin two-hybrid assays, Nakashima et al. [9] have shown that full-length OsR-bohB did not interact with OsRACK1A, and the author hypothesized that the C-terminus of RbohB has an inhibitory effect on the interaction. The reports indicated that among all isoforms, respiratory burst oxidase homologue D (RbohD) is the most active and highly expressed for apoplastic ROS generation [52,54]. We also found elevated RbohD transcription in OsRACK1B-OX plants ( Figure 3D). Therefore, to further verify our observation, we deliberately chose the N-terminal region of OsRbohD encompassing amino acid 1-410, which was cloned with the C-terminal part of YFP, and the full-length OsRACK1B was fused with both the N-and the C-terminal part of YFP for our BiFC experiment. We observed fluorescence in the cytoplasm indicative of protein-protein interactions in normal non-stressed onion cells bombarded with OsRACK1B-nYFP and OsRbohD (1-410)-cYFP constructs in the BiFC assays (Figure 4, top panel and Supplemental Figure S4A). To examine whether the expression of both OsRACK1B and OsRbohD can be induced by environmental stimuli such as NaCl treatment, the epidermal cells were incubated with Murashige and Skoog medium containing 200 mM NaCl for 30 min. Indeed, a strong YFP fluorescence was detected in the nucleus of the plasmolyzed cells (Figure 4, second row and Supplemental Figure S4B), suggesting that OsRACK1B interacts with the OsRbohD in the cytoplasm in a physiological condition, but the complex moves into the nucleus in response to environmental stimuli. The trigger for such shuttling between the cytoplasm and nucleus, however, remains to be determined. In the control, the coexpression of OsRACK1B and OsRbohD (1-410 aa) constructs showed DAPI fluorescence but no YFP signals (bottom two panels). The onion epidermal cells were transiently transformed with the full-length OsRACK1B cds and N-terminal region of OsRbohD consisting of 1 to 410 amino acid sequences. Blue fluorescence from DAPI (4 9, 6-diamidino-2-phenylindole) staining indicates nuclei, merge, merged images of YFP channel, DAPI, and differential interference contrast image (DIC). Constructs were fused to either carboxy (YFP-C) or amino (YFP-N) terminus of YFP and vice versa. All constructs were under the control of the 35S promoter. Each pair of recombinant plasmids encoding nYFP and cYFP fusions was mixed 1:1 (w/w) and co-bombarded into onion epidermal cell layers. The transformed onion epidermal layers were incubated at 22 °C for 16-24 h. For plasmolysis, epidermal cells were incubated in liquid MS media containing 200 mM NaCl for 30 min following bombardment to separate cytoplasm from cell walls. The coexpression of nYFP with OsRbohD (1-410 aa)-cYFP serves as a negative control as there are no YFP signals observed. Bars = 100 μm.

Overexpression of OsRACK1B Affects Fertility, Anther Dehiscence, and Spikelet Fertility
It is known that RACK1 is involved in multiple developmental processes, including plant growth, leaf production, and flowering [1,4]. In our study, one of the most pronounced features of the OsRACK1B-OX line was that the plants displayed reduced fertility with shorter floral organs, including panicles, spikelets, palea, lemma, and anthers, than those of the wild-type plants. To further characterize the role of RACK1B in rice, we then investigated the fertility rate and pollen development of the OsRACK1B-OX transgenic rice plants. We examined the flowering and fertility phenotypes of OsRACK1B-OX and WT plants. The microscopic observation of the female reproductive organs appeared normal. Therefore, to investigate whether the reduced fertility was due to pollen viability, we used KI-I2 staining which reflects the amount of starch accumulated in the pollen and is highly correlated with pollen fertility [55,56]. Starch provides energy for pollen maturation and pollen tube germination [57]. In general, viable pollens are stained a dark purple color due to enough starch presence, whereas those lightly stained or unstained are In the control, the coexpression of OsRACK1B and OsRbohD (1-410 aa) constructs showed DAPI fluorescence but no YFP signals (bottom two panels). The onion epidermal cells were transiently transformed with the full-length OsRACK1B cds and N-terminal region of OsRbohD consisting of 1 to 410 amino acid sequences. Blue fluorescence from DAPI (4 9, 6-diamidino-2-phenylindole) staining indicates nuclei, merge, merged images of YFP channel, DAPI, and differential interference contrast image (DIC). Constructs were fused to either carboxy (YFP-C) or amino (YFP-N) terminus of YFP and vice versa. All constructs were under the control of the 35S promoter. Each pair of recombinant plasmids encoding nYFP and cYFP fusions was mixed 1:1 (w/w) and co-bombarded into onion epidermal cell layers. The transformed onion epidermal layers were incubated at 22 • C for 16-24 h. For plasmolysis, epidermal cells were incubated in liquid MS media containing 200 mM NaCl for 30 min following bombardment to separate cytoplasm from cell walls. The coexpression of nYFP with OsRbohD (1-410 aa)-cYFP serves as a negative control as there are no YFP signals observed. Bars = 100 µm.

Overexpression of OsRACK1B Affects Fertility, Anther Dehiscence, and Spikelet Fertility
It is known that RACK1 is involved in multiple developmental processes, including plant growth, leaf production, and flowering [1,4]. In our study, one of the most pronounced features of the OsRACK1B-OX line was that the plants displayed reduced fertility with shorter floral organs, including panicles, spikelets, palea, lemma, and anthers, than those of the wild-type plants. To further characterize the role of RACK1B in rice, we then investigated the fertility rate and pollen development of the OsRACK1B-OX transgenic rice plants. We examined the flowering and fertility phenotypes of OsRACK1B-OX and WT plants. The microscopic observation of the female reproductive organs appeared normal. Therefore, to investigate whether the reduced fertility was due to pollen viability, we used KI-I 2 staining which reflects the amount of starch accumulated in the pollen and is highly correlated with pollen fertility [55,56]. Starch provides energy for pollen maturation and pollen tube germination [57]. In general, viable pollens are stained a dark purple color due to enough starch presence, whereas those lightly stained or unstained are considered non-viable. We found that approximately 20-40% of the pollens from the OsRACK1B-OX plants were darkly stained, compared with >80% of the pollens from the wild-type ( Figure 5A,C,E,L). As shown in Figure 5G,I,N, the seed-setting rate of the OsRACK1B-OX plants was only 20-30%, nearly a quarter that of the wild-type (85%), which is consistent with abnormal pollen morphology. To gain further insights, we performed a detailed morphological analysis of the anthers at the flowering stage of the transgenic rice plants. A semi-thin transverse section using toluidine blue staining revealed that OsRACK1B-OX plants had a shrinking anther and a smaller pollen sac than that of the wild-type anthers ( Figure 5B,D,F). In addition, the number of pollen grains per locule was only 10-20 grains per locule in RACK1B-OX anthers, less than half that of the wild-type (40 grains per locule) ( Figure 5M). We also observed abnormality in anther dehiscence and flower opening in the OsRACK1B-OX plants. In the wild-type, the anthers dehisced completely and shed pollen before the spikelet began to close ( Figure 5H). After complete dehiscence of the anthers, the spikelet closes, keeping the empty anthers outside the spikelet. However, we observed that most of the spikelets of the OsRACK1B-OX plants remained open during anthesis, and the anthers remained indehiscent ( Figure 5J). This observation suggested that the reduced spikelet fertility is due, at least in part, to the inability of the anthers to dehisce, the low viability of the pollen grains, and defects in spikelet closing. Combinedly, our analysis implied that OsRACK1B plays a critical role in panicle development and anther dehiscence in rice. The pollen wall consists of several layers: exine in the outer layer, which includes tectum; bacula links exine with the inner layer intine, which includes nexine, the pollen coat, and the foot layer [58][59][60][61]. The intine development is similar to that of plant cells and is made of celluloses, pectin, hydrolytic enzymes, and hydrophobic proteins, required to maintain the structural integrity of the pollen grains, pollen maturation, and pollen tube growth for fertility [60,[62][63][64]. Higher magnification TEM images revealed defective intine development in the OsRACK1B-OX pollens compared with that in the wild-type. More specifically, the OX grains accumulated a thicker intine layer (0.86 µ m in OX-1 and 1.28 µ m in OX-2) compared with that of the wild-type pollen grain (0.72 µ m) ( Figure 6D-F). It is hypothesized that abnormalities in intine formation lead to the arrest of pollen development at the maturation stage, resulting in a lack of starch granule accumulation [62,65,66]. These differences are also in agreement with our KI-I2 staining and thin-section analysis of wild-type and mutant pollen.

OsRACK1B-OX Plants Exhibit Defective Pollen Morphology and Delayed Dehiscence
Generally, anther dehiscing starts when the anthers reach the top of the spikelet and release pollen grain over the stigma right before the spikelet opening. After anthesis, the filaments elongate further. As soon as the remaining pollen grains are released, the spikelet closes, keeping the empty anthers outside the spikelet ( Figure 5H). Unlike the wild-type plant, the anthers of the RACK1B-OX plants did not dehisce at the time of spikelet opening ( Figure 5J). While other the reproductive organs appeared normal, we assumed that the spikelet sterility of the RACK1B-OX plants was due mainly to the inability of the anthers to dehisce and release pollen grains normally.
To better understand the structure of the defective pollens during pollen development, we used transmission electron microscopy (TEM) analysis for the structure of the pollen grains from both the wild-type and the OsRACK1B-OX plants. At the mature stage, the most obvious difference between pollen sources was in the number of starch granules inside the pollens. Whereas the wild-type pollen grain had accumulated abundant starch granules ( Figure 6A), the OsRACK1B-OX pollen grains had decreased starch granules with irregular shapes (Figure 6B,C). This observation was corroborated with our KI-I 2 staining experiment. Other apparent abnormalities were in the vacuole size, being smaller in the wild-type ( Figure 6A,D), but remaining much larger in the OX lines ( Figure 6B,C,E,F). In addition, abnormality of the cytoplasm density was apparent in the OsRACK1B-OX pollens ( Figure 6E,F) compared to that of the wild-type ( Figure 6D). Interestingly, DAPI staining of aborted pollens from the anthers that showed indehiscence or delayed dehiscence revealed that almost all the pollen grains (from OsRACK1B-OX) contained three, two, one, or no DAPI-stained nuclei in intact or completely collapsed forms with callose patches on the pollen wall (Supplemental Figure S5A,B).

Discussion
As a scaffold protein, RACK1A has been widely studied and has been implicated in plant growth, development, and stress response pathways, but the functional characterization of RACK1B remains to be explored. In addition, RACK1′s functional role in cereal crops such as rice is still in its infancy. Over the past decades, an increasing number of studies have elucidated RACK1′s contribution to seed germination and defense response mechanisms via ROS signaling cascades [9,10,39,67,68]. Nevertheless, the impact of the overlapping RACK1 and ROS signaling pathways in the context of rice pollen development and fertility have not been examined yet. In this study, we carried out a complex characterization of OsRACK1B through biochemical, histochemical, and ultrastructure analysis. The pollen wall consists of several layers: exine in the outer layer, which includes tectum; bacula links exine with the inner layer intine, which includes nexine, the pollen coat, and the foot layer [58][59][60][61]. The intine development is similar to that of plant cells and is made of celluloses, pectin, hydrolytic enzymes, and hydrophobic proteins, required to maintain the structural integrity of the pollen grains, pollen maturation, and pollen tube growth for fertility [60,[62][63][64]. Higher magnification TEM images revealed defective intine development in the OsRACK1B-OX pollens compared with that in the wild-type. More specifically, the OX grains accumulated a thicker intine layer (0.86 µm in OX-1 and 1.28 µm in OX-2) compared with that of the wild-type pollen grain (0.72 µm) ( Figure 6D-F). It is hypothesized that abnormalities in intine formation lead to the arrest of pollen development at the maturation stage, resulting in a lack of starch granule accumulation [62,65,66]. These differences are also in agreement with our KI-I 2 staining and thin-section analysis of wildtype and mutant pollen.

Discussion
As a scaffold protein, RACK1A has been widely studied and has been implicated in plant growth, development, and stress response pathways, but the functional characterization of RACK1B remains to be explored. In addition, RACK1 s functional role in cereal crops such as rice is still in its infancy. Over the past decades, an increasing number of studies have elucidated RACK1 s contribution to seed germination and defense response mechanisms via ROS signaling cascades [9,10,39,67,68]. Nevertheless, the impact of the overlapping RACK1 and ROS signaling pathways in the context of rice pollen development and fertility have not been examined yet. In this study, we carried out a complex characterization of OsRACK1B through biochemical, histochemical, and ultrastructure analysis.
Emerging evidence suggests that a certain threshold level of ROS, such as H 2 O 2 , can initiate intracellular signaling cascades associated with responses to abiotic and biotic stressors in living organisms [28,31,32,[69][70][71][72][73][74][75]. ROS generated inside the cell converts H 2 O 2 by the dismutation of O 2 , either spontaneously or via superoxide dismutase (SOD) catalysis [76,77]. The mobility, selectivity, and relative stability of H 2 O 2 make it an ideal molecule to act as a second messenger that transduces retrograde signals from chloroplasts to the nucleus in response to stress [78,79]. While chloroplast, mitochondria, and peroxisomes are considered the major source of metabolic ROS, the reports suggest apoplasts as a site for signaling ROS generation that can cross-talk with other sources in response to stress adaptation [27,80]. The reports indicate that OsRACK1 directly interacts with the N-terminus of the NADPH oxidase (Rboh) and stimulates ROS generation in rice [9]. In agreement with previous findings, we also showed that OsRACK1B interacts with the N-terminal end of RBOH. AtRACK1 was also found to bind CuZn-SOD in split-ubiquitin-based yeast-twohybrid assays [81]. Considering the intracellular mobility of H 2 O 2 [82,83], it is tempting to speculate that ectopic expression of RACK1B-activated PM-bound Rboh generates H 2 O 2 via the action of apoplastic CuZn-SODs, resulting in further biosynthesis of ROS as a systemic wave and generating a positive feedback loop for homeostasis. However, the exact mechanism needs further investigation.
Accumulating evidence suggests that, in contrast to intracellular sources, membranelocalized or apoplastic ROS (in particular H 2 O 2 ) can act as a signaling molecule at a threshold level to regulate growth and development and adaptation to various biotic and abiotic stresses [73,[84][85][86][87][88]. H 2 O 2 is recognized as the central molecule in redox signaling due to its long lifespan, low reactivity, membrane permeability, mobility from its generation site to a target site, and stability compared to singlet O 2 [71,89]. Under normal physiological conditions, H 2 O 2 production is tightly controlled and fine-tuned by ROS scavenging enzymes and low molecular weight components. RBOH/NADPH oxidase is known as the major source of ROS, presumably H 2 O 2 , in plant cells. Structural analysis suggests that RBOH consists of six transmembrane domains, NADPH hydrophilic domains, two N-terminal Ca 2+ -binding EF-hand motifs that include a GTPase binding region, a C-terminal dehydrogenase domain that binds flavin adenine dinucleotide (FAD), and a functional oxidase domain responsible for superoxide production by transferring electrons from NADPH to oxygen [51,90]. Studies in different plant species have shown that post-transcriptional or post-translational regulation is crucial for Rboh activity [15,37,46,52]. The N-terminal region undergoes post-translational modification by several protein kinases, such as calcium-dependent protein kinases (CDPKs) and calcineurin B-like proteininteracting protein kinases (CIPKs), or Ca + -independent kinases, such as receptor-like cytoplasmic kinases (RLCKs), plasma-membrane-associated kinase BIK1 (Botrytis-induced kinase1), and mitogen-activated protein kinases (MAPKs) [37,46,91]. Interestingly, Rboh activity can also be regulated by its interacting partners, such as OsRACK1 [9] and 14-3-3 proteins [92]. Our experiment suggests that OsRACK1B binds to the N-terminus cytoplasmic region of the RbohD. Therefore, we speculate that this binding makes it susceptible to post-translational modification, and OsRboh in OsRACK1B-OX lines has more opportunity to bind and allow post-translational modification by other proteins as well as allow the ROS generation. We also hypothesize that the RBOH enzyme is undergoing a post-translational modification and may contain different or additional phosphorylation or sumolyation subunits that are responsible for this shift in its enzymatic activity [30,37,93,94].
In our BiFC analysis, we observed that OsRACK1B and OsRbohD interact in the cytoplasm in onion epidermal cells, which is expected given that both proteins were reported to be localized in the cytoplasm in plants [4,9,95]. Intriguingly, the complex translocates to the nucleus in response to salt stress, possibly as a part of a signal transduction pathway to regulate oxidative stress-related gene expression. Transcriptional reprogramming is a hallmark strategy of a defense response in the living system. Oxidative stress such as high salinity provokes large-scale changes in the transcriptomic landscape and generates a systemic ROS wave [27,32,36,96,97]. Plants integrate multiple signals from different cellular compartments to adapt to stress conditions. Retrograde signaling from plastid, mitochondria, cytoplasm, or chloroplast to the nucleus to express a specific set of genes is a common intracellular stress signaling system in a fluctuating environment [28,74,98]. Apoplastic ROS, such as H 2 O 2 itself as a signaling molecule, instigates a change in gene expression in the nucleus. For instance, the nonexpressor of pathogenesis-related genes 1 (NPR1) protein, a redox-sensitive transcription coactivator, primarily localized in the cytoplasm in both Arabidopsis and tobacco, transported to the nucleus in response to salt stress to function as a coactivator of gene transcription [99][100][101]. Recently, it was shown that a nuclear translocation of GmMPK6 from the cell membrane during high salinity treatment in soybean seedlings can be blocked by DPI treatment, suggesting that the nuclear translocation is mediated by Rboh-dependent H 2 O 2 [102]. In this aspect, it is unclear whether the OsRACK1B-OsRbohD complex itself moves to the nucleus for gene regulation or H 2 O 2 produced in response to salt treatment triggers the translocation. Further research will provide more insights on which mechanism the nuclear translocation is mediated in response to oxidative stress.
Most of the studies on Rboh-dependent ROS production have focused on its contribution to the biotic and abiotic stress tolerance in plants. RBOH has also gained attention for its role in seed development, germination, flowering, and other reproductive organs, such as pollens and stamens [20,21,[103][104][105].
ROS, in particular H 2 O 2 , has shown to be involved in pollen grain activation in kiwifruit [106,107], hyperpolarization of lily, and tobacco pollen grain protoplast [108][109][110]. Several reports suggest that a tightly controlled spatio-temporal regulation of ROS homeostasis is crucial for pollen development. The reports indicated that excessive accumulation of H 2 O 2 is responsible for male sterility in rice [23,60,111,112]. During the maturation stages of pollen development, a process called program cell death (PCD) of the tapetum (the highly specialized innermost layer of anther) is essential for pollen development [113,114]. The reports showed that PCD of the Arabidopsis and rice anther tapetum is induced by ROS and leads to the loosening of the pollen grains from surrounding tissues and the development of pollen materials and coatings [61,111]. ROS contributes to the PCD and tapetum degeneration process of pollen development in a time-dependent manner [114]. Therefore, the abnormalities we observed in the pollens in the OsRACK1B-OX plants in our study may be associated with the constitutive accumulation of OsRACK1B-induced H 2 O 2 , leading to the premature initiation of PCD in pollen grains and pollen abortion. We also hypothesize that the defective intine structure is linked to the over-accumulation of H 2 O 2 as similar findings were observed by Smirnova et al. [106,115]. The author demonstrated that H 2 O 2 regulates tobacco pollen germination by modifying the pollen wall properties. They also suggest that H 2 O 2 is responsible for the thickening of the cross-linking structure of the intine layer. It would be interesting to dissect the detailed regulatory mechanisms of how RACK1B contributes to the anther development process.
Pollen wall thickness directly influences pollen longevity [59,116]. The rice pollen wall is generally thin and short-lived compared to other grass species. Rice produces recalcitrant pollen, which remains viable for only a few minutes after anthesis [56,117]. The thin pollen wall in rice facilitates both fast germination on the stigma and dehydration in the air, leading to short longevity [117]. Studies related to pollen wall formation confirmed that pollen wall formation is tightly linked with anthesis, panicle development, and spikelet fertility [118][119][120]. The reduced fertility of the RACK1B-OX plants observed in our study can be explained by the defective architecture of the pollen wall. Indeed, mutation studies on genes important for another development have demonstrated that lack of starch granule accumulation and abnormal intine thickness resulted in male sterility in rice [120][121][122][123]. Our ultrastructure observation suggests that OsRACK1B may also play a direct or indirect role in pollen structure development. However, further studies are necessary to explore the detailed mechanism.
Seeds were surface sterilized and germinated on full-strength Murashige and Skoog (MS) [127] culture media (Caisson Laboratories, Inc.) at room temperature. Leaves from two-week-old germinated seedlings were used for DNA extraction and genotyping. Wildtype (WT) and selected transgenic lines were then transferred to a hydroponic nutrient solution prepared following the protocol as described by Lakshmanan et al. [128]. Plants were grown at 28 • C during a 14 h light (300 µmol m −2 s −1 ) period and at 24 • C during 10 h of darkness with 60% relative humidity. Leaf tissues from the plants were collected, flash-frozen in liquid nitrogen, and stored at −80 • C for protein and RNA extraction.  Figure S1), as described by Jeong et al. [125,126]. The LP and RP primers were also used for detecting the OsRACK1B gene.
The insertion site of the T-DNA in the transgenic lines was mapped by Thermal Asymmetric Interlaced (TAIL) PCR of the genomic DNA according to previously described protocols (Supplemental Figure S2 and Methods S1) [129]. The PCR products were Sanger sequenced and analyzed using the NCBI BLAST service. All of the primers used for genotyping and TAIL-PCR procedures are listed in Tables S1 and S3.

RNA Extraction, Complementary DNA (cDNA) Synthesis, and Quantitative Real-Time PCR (qRT-PCR) Analysis
Total RNA was extracted from a pool of eight-week-old rice leaves from each plant and reverse transcribed to make cDNA using the SuperScript IV VILO Master Mix kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions.
Quantitative real-time PCR (qRT-PCR) was performed using cDNA and the PowerUP SYBR Green master mix (Thermo Fisher Scientific) with a CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA). Normalized expression (2(-Delta Delta C(T) method) was calculated using the Bio-Rad CFX manager software, employing the housekeeping gene OsActin1 (Os03g50885) as a reference gene. All experiments were performed in triplicate for technical repeats. The results were plotted as relative values ±SEM and graphically displayed using GraphPad Prism version 8.2.0 (GraphPad Software, Inc., San Diego, CA, USA). The qRT-PCR for each gene was repeated three times. The primer sequences used are listed in Table S2.

Protein Extraction and Western Blot Analysis
Total protein from the leaves was extracted from 100 mg of finely powdered tissue using lysis buffer (CelLytic P, Sigma-Aldrich, St. Louis, MO, USA) containing a protease and phosphatase inhibitor cocktail (Sigma-Aldrich). Total protein content for each sample was quantified by Bradford assay using the Quick Start Bradford dye reagent (Bio-Rad).
Western blot analysis was performed using an equal amount of total proteins per sample and resolved in 4-12% pre-cast XT-MES gel (Bio-Rad). The proteins were transferred to polyvinylidene difluoride (PVDF) membrane and incubated with primary and secondary antibodies. Membranes were developed using the ECL detection kit (Bio-Rad). The signals of the bands were visualized and captured using the Chemi-DocXRS system (Bio-Rad). The OsRACK1B peptide-specific mAb antibodies were raised using two peptides, AGVL-RGHNDM and QDLKPEVQAF, corresponding to amino acids 10-19 at the N-terminus and 286-295 at the C-terminus end of the OsRACK1B protein sequences, respectively (Abmart, Shanghai, China). The anti-RBOHD (cat# AS15 2962, sample for validation from Agrisera, Sweden) antibody used in this study demonstrated reactivity as stated on the manufacturer's website. Ponceau-S (40% methanol (v/v), 15% acetic acid (v/v), and 0.25% Ponceau-S) staining of Rubisco was used as the loading control.

Histochemical Staining of Reactive Oxygen Species (ROS)
Diaminobenzidine (DAB) and 2 ,7 -dichlorodihydrofluorescein diacetate (H 2 DCFDA) stains were used to detect the in situ H 2 O 2 level in leaves and roots. The stained tissues were evaluated using compound and spinning disk confocal system microscopes.
The DAB assay was conducted according to the protocol described by Daudi and O'Brien [130]. Briefly, leaves (fully expanded) from eight-week-old wild-type and transgenic plants were excised and floated in the water for an hour to minimize wound-induced ROS production. The samples were vacuum infiltrated in 1 mg/mL DAB-HCL (Sigma-Aldrich, St. Louis, MO, USA) solution for 5 min, followed by incubation in DAB solution for 48 h in darkness at room temperature. Following incubation, the reaction was stopped by washing the leaves with a clearing solution (3:1, 95% ethanol: glacial acetic acid). Chlorophyll from the leaves was removed by boiling with a clearing solution in a six-well plate placed in a water bath (95 • C) for 20 min and photographed using a Leica EZ4 stereo-microscope.
Eight-week-old wild-type and transgenic plant leaves and roots were used for an H 2 DCFDA experiment, as described by Achard et al. [131]. Briefly, the samples were

In-Gel Activity Assay of Rboh/NADPH and SOD Enzymes
Native polyacrylamide gel electrophoresis (N-PAGE) was carried out using 4-15% Mini-PROTEAN TGX Stain-Free Gels and native sample buffer (both from Bio-Rad, Hercules, CA, USA) in the Laemmli (1970) buffer system without sodium dodecyl sulfate (SDS). The gels were first run at 20 mA for one hour, after which the electrophoresis buffer was replaced. The gels were then loaded with an equal amount of protein sample and run at 40 mA for 2-3 h to the run-off of the dye front.
Rboh/NADPH native gel assay was performed according to the protocol described by Sagi and Fluhr [104]. For this assay, a plasma membrane (PM) fraction of the leaf extracts was prepared. Freshly harvested leaves were homogenized using homogenization buffer (50 mM HEPES pH 7.5, 0.4 M sucrose, 0.1 M KCl, 0.1 M MgCl 2 , and plant protease inhibitor cocktail (#P9599; Sigma) in the ratio of 1:3 (w/v) and immediately preserved at 4 • C. The homogenate was centrifuged at 10,000× g for 10 min. The supernatant was filtered through Miracloth (Millipore). The microsomal membranes (MF fraction) were separated from the cytosolic fraction (CF) by centrifugation at 30,000× g for 60 min at 4 • C. Pellets (MF) were resuspended in 5 mM phosphate buffer, pH 7.8. The NADPH-dependent superoxide anion radical (O 2 •-) producing capabilities of the membrane fractions were assayed in gels by a modified nitroblue tetrazolium (NBT) reduction method (). Briefly, 20 µg MF protein samples were electrophoresed on a native polyacrylamide gel (4-15% Mini-PROTEAN TGX, Bio-Rad, Hercules, CA, USA), and the gel was incubated in the dark for 20 min in a reaction mixture solution containing 50 mM Tris-HCl buffer (pH 7.5), 0.4 mM NBT (Sigma-Aldrich), 0.1 mM MgCl 2 , and 2.5 mM CaCl 2 . After incubation, 134 µM NADPH was added, and the appearance of blue formazan bands was monitored. The reaction was stopped by immersion of the gels in distilled water and photographed immediately using a flatbed scanner. For control, 50 µM DPI was added to the specific well.
MnSOD and CuZn-SOD native gel activity assay was performed according to the protocol described by Weydert and Cullen [132], with slight modifications. Briefly, the gel was incubated in 2.43 mM NBT for 20 min, followed by soaking in 50 mM phosphate buffer of pH 7.8, containing 28 mM TEMED (Sigma-Aldrich) and 2.8 mM riboflavin for 15 min. The SOD isozymes were visualized as colorless bands as the gel turned blue following exposure to white cool fluorescent illumination until the gel turned blue/purple, and clear achromatic bands appeared. The SOD isozymes were identified using inhibitors, 8 mM H 2 O 2 , and 2 mM KCN. The gels were scanned, and negative images were obtained.

Bimolecular Fluorescence Complementation (BiFC) Assay
For the BiFC assay, full-length OsRACK1B and 1300 bp of RBOHD coding sequences of the N-terminal region were recombined in Gateway-compatible BiFC vectors [133]. Each pair of recombinant plasmids encoding nEYFP or cEYFP fusion proteins was co-bombarded into onion epidermal cells using the Helios DNA particle delivery system (Biolistic PDS-1000/He, BioRad), as described by Hollender and Liu [134]. The onion cells were incubated in MS liquid media for 16-24 h at 22 • C under dark incubation and observed for YFP fluorescence with an inverted spinning-disk confocal microscope (Eclipse Ti-E-PFS, Nikon, Melville, NY, USA). For salt treatment, the onion cells were incubated in liquid MS media with 200 mM NaCl for 30 min before imaging. Confocal fluorescent and DIC images were acquired and processed using the Nikon NIS-Elements software. Negative controls for interaction were provided by empty YFPC and YFPN vectors in combination with the OsRACK1B and RBOHD pSAT expression plasmids (Methods S2) (133). The primers used for BiFC plasmids are listed in Table S4.

Phenotypic Characterization, Pollen Viability Assay, and DAPI Staining
To analyze pollen viability, anthers from mature flowers were dissected before anthesis and fixed in 70% ethanol, and the pollen grains were stained with I 2 -KI staining (0.2% iodine and 2% potassium iodide), as described by Zhou et al. [135]. The stained pollen grains were then photographed with a Leica EZ4 HD microscope. The total number of pollen grains and viable pollen were counted with the software ImageJ (http://rsbweb.nih.gov/ij/, accessed on 12 May 2019). The pollen grains densely stained by the I 2 -KI solution were counted as mature viable pollen.
For 4 ,6-diamidino-2-phenylindole (DAPI), single anthers were dissected from the isolated spikelet using a dissecting microscope (Leica EZ4 HD). Mature anthers from the WT and transgenic plants were disrupted on microscope slides using dissecting needles and gently squashed in DAPI staining solution (VECTASHIELD Antifade Mounting Medium with DAPI, Newark, CA, USA) under a cover slip. After staining for 20 min, the fluorescent images were acquired using a Nikon Ti-E-PFS inverted spinning-disk confocal microscope. Photos of the panicles were taken using a point-and-shoot digital camera. Fresh rice spikelets, flowers, and dehiscent anthers were photographed with a Leica EZ4 HD stereo microscope.

TEM and Cross-Section Analysis of Pollen
For transmission electron microscopic (TEM) and cross-section analysis, anthers at the maturation stage from wild-type and RACK1B-OX plants were fixed by immersion in 2% paraformaldehyde-glutaraldehyde fixative in 0.1 M sodium cacodylate buffer (pH 7.2) for 1 h at room temperature. Specimens were rinsed in the same buffer and post-fixed in 2% osmium tetroxide for 3 h, dehydrated in a graded series of ethanol to 100%, and embedded in epoxy resin. For SEM, the anther specimens were thin-sectioned (2-3 µm) with a microtome (LKB Ultratome V; LKB) and stained in 1% toluidine blue. Bright-field photographs of the cross-sections were taken using a Keyence VHX 6000 digital microscope.
For TEM, ultrathin sections (70 to 80 nm) were made with an LKB-2088 ULTROTOME V (LKB) using a diamond knife and mounted on 200-mesh copper grids. The sections were stained with 2% uranyl acetate and lead citrate and observed with a Hitachi S-4700 electron microscope with a transmitted electron detector operating at 80 kV.

Conclusions
In this study, we found that the overexpression of OsRACK1B triggers RBOH-dependent H 2 O 2 production, leading to a systemic ROS wave in the whole plant. We also found that fertility and pollen development is severely impaired in OsRACK1B-OX plants. Based on our data, we propose that the OsRACK1B-induced ROS wave spread to the distal plant cells, leading to a change in the redox status during a highly sensitive pollen development process, ultimately affecting the fertility and seed settings in rice.
The importance of pollen development in crop yields is enormous; so, understanding the role of the RACK1-mediated pollen development pathway is very important from the scientific as well as the economic points of view. Being sessile organisms and under the global climate change scenario, plants must adapt to environmental stresses through a highly balanced and finely tuned interaction between signaling molecules. Similarly, successful reproductive growth is important for crop productivity, and various defects in reproductive organ development, such as male sterility, cause adverse crop yields. Understanding the molecular basis of ROS regulation and yield biology is the key to achieving sustainable productivity of climate-sensitive cereal crops such as rice.


Development of p-Tau Differentiated Cell Model of Alzheimer’s Disease to Screen Novel Acetylcholinesterase Inhibitors

Alzheimer’s disease (AD) is characterized by an initial accumulation of amyloid plaques and neurofibrillary tangles, along with the depletion of cholinergic markers. The currently available therapies for AD do not present any disease-modifying effects, with the available in vitro platforms to study either AD drug candidates or basic biology not fully recapitulating the main features of the disease or being extremely costly, such as iPSC-derived neurons. In the present work, we developed and validated a novel cell-based AD model featuring Tau hyperphosphorylation and degenerative neuronal morphology. Using the model, we evaluated the efficacy of three different groups of newly synthesized acetylcholinesterase (AChE) inhibitors, along with a new dual acetylcholinesterase/glycogen synthase kinase 3 inhibitor, as potential AD treatment on differentiated SH-SY5Y cells treated with glyceraldehyde to induce Tau hyperphosphorylation, and subsequently neurite degeneration and cell death. Testing of such compounds on the newly developed model revealed an overall improvement of the induced defects by inhibition of AChE alone, showing a reduction of S396 aberrant phosphorylation along with a moderate amelioration of the neuron-like morphology. Finally, simultaneous AChE/GSK3 inhibition further enhanced the limited effects observed by AChE inhibition alone, resulting in an improvement of all the key parameters, such as cell viability, morphology, and Tau abnormal phosphorylation.

Introduction
Alzheimer's disease (AD) is a neurodegenerative disease, currently recognized as the worldwide leading cause of dementia, reportedly affecting more than 50 million patients in the world, with this figure expected to be at least doubled by 2050 [1]. The disease is characterized by an array of cognitive symptoms, such as memory impairment, learning disabilities, and behavioral changes, which lead to a progressive loss of independency in routine tasks by the affected patients [2]. The typical pathology is presented by a severe progressive neurodegeneration, accumulation of amyloid plaques, and presence of neurofibrillary tangles (NFTs) [3].
NFTs are found in the neuronal cytoplasm of AD patients and are composed of abnormally phosphorylated Tau protein. Tau hyperphosphorylation is the result of the aberrant activation of either kinases or phosphatases. One of the most studied kinases, responsible for the phosphorylation on Tau protein at multiple residues, is GSK3-β. The latter is ubiquitously expressed in the central nervous system (CNS), and is activated by a number of different pathways, which are often linked to AD [4][5][6][7][8]. Moreover, phosphorylation performed by GSK-3-β on Tau results in a loss of affinity to microtubules and promotion of Tau self-aggregation [9,10]. In AD, as well as in other tauopathies, the aberrant phosphorylation process that occurs on Tau triggers its assembly in paired helical filaments (PHF) and straight filaments (SF), which leads to the development of NFTs [11].
It has been also shown that hyperphosphorylated Tau is required to trigger amyloid toxicity in vivo [12,13].
Multiple factors are involved in the triggering of Tau hyperphosphorylation, such as amyloid plaques, glucose metabolism impairment, and RAGE-pathway activation [14][15][16]. Despite being through different pathways, all factors involved in altering Tau phosphorylation levels lead to the activation of specific kinases enzymes or the inhibition of phosphatases enzymes [17].
In the present study, we have focused on GSK3-β as a potential target for AD therapy, which is responsible for phosphorylating Tau on residue S396, in which abnormal phosphorylation is considered to be an early marker of subsequent hyperphosphorylation [18]. GSK3-β is the downstream kinase activated by multiple pathways linked to Tau hyperphosphorylation [5,[18][19][20][21]. Thus, targeting GSK3-β could result in the inhibition of Tau phosphorylation induced by multiple pathways in AD, and, therefore, block the disease progression.
The complexity of AD has frequently limited the realism of experimental models, which often results in contradictive results. Cell line SH-SY5Y has often been used as a cell culture model of AD, in particular for evaluating AChE inhibitors due to its expression of AChE enzyme in cells [22][23][24][25], despite bearing the crucial limitation of being constantly dividing. However, several approaches have been proposed to further improve the SH-SY5Y cell model by using retinoic acid (RA) as a differentiation factor, and foetal bovine serum (FBS) deprivation to stop the constant division [26][27][28][29][30]. Moreover, a number of methods exploiting undifferentiated SH-SY5Y modelled Tau hyperphosphorylation through different mechanisms, such as hypothermia, okadaic acid administration, and glyceraldehyde (GA) exposure [31][32][33]. The usage of GA to induce Tau hyperphosphorylation is of peculiar interest, since GA treatment leads to increased levels of advanced glycated end products (AGEs), and the receptor, RAGE, activates an important and well-studied pathway in GSK3-β activation [20,21,33].
To study hyperphosphorylation mechanisms of Tau we have developed and validated a new cell-based AD model by differentiating SH-SY5Y into mature neurons featuring morphological and p-Tau characteristics with administration of GA to trigger the Tau aberrant phosphorylation [26,33]. Finally, we exploited such a model to assess the efficacy of novel single-or dual-targeting acetylcholinesterase (AChE) inhibitors; the core structure of these new drug candidates is derived from the (±)-7,8-Dihydroxy-3-methylisochroman-4-one [(±)-XJP] compound, which was initially isolated from the Musa sapientum L., with a 4-F as R group and named XJP-1 [34][35][36]. In addition to this, given the relevance and central role of the GSK3-β enzyme in multiple pathways during AD pathogenesis, a novel dual AChE/GSK3-β inhibitor named 27g has been tested (Table 1) [37].

GA Treatment Induces Abnormal Tau Phosphorylation and Axon Degeneration in SH-SY5Y-Differentiated Neurons
To develop a neuron-like model of AD bearing Tau hyperphosphorylation, the d ferentiation of SH-SY5Y to neurons was firstly performed as described by Shipley et [26]. SH-SY5Y-derived neurons were subsequently imaged by confocal microscopy (F ure 1A,B) to compare morphological differences. Differentiated neurons exhibited a s nificant increment in neurite density and neurite length when compared to undifferen ated SH-SY5Y cells ( Figure 1A,B).
After validating the neuronal differentiation of SH-SY5Y cells, we investigat whether GA treatment for 24 h would result in any AD defects in differentiated neuro as described by Koriyama in undifferentiated SH-SY5Y cells [33]. Confocal imaging, a Name:

GA Treatment Induces Abnormal Tau Phosphorylation and Axon Degeneration in SH-SY5Y-Differentiated Neurons
To develop a neuron-like model of AD bearing Tau hyperphosphorylation, the d ferentiation of SH-SY5Y to neurons was firstly performed as described by Shipley et [26]. SH-SY5Y-derived neurons were subsequently imaged by confocal microscopy (F ure 1A,B) to compare morphological differences. Differentiated neurons exhibited a s nificant increment in neurite density and neurite length when compared to undifferen ated SH-SY5Y cells ( Figure 1A,B).
After validating the neuronal differentiation of SH-SY5Y cells, we investigat whether GA treatment for 24 h would result in any AD defects in differentiated neuro as described by Koriyama in undifferentiated SH-SY5Y cells [33]. Confocal imaging, a Name:

GA Treatment Induces Abnormal Tau Phosphorylation and Axon Degeneration in SH-SY5Y-Differentiated Neurons
To develop a neuron-like model of AD bearing Tau hyperphosphorylation, the d ferentiation of SH-SY5Y to neurons was firstly performed as described by Shipley et [26]. SH-SY5Y-derived neurons were subsequently imaged by confocal microscopy (F ure 1A,B) to compare morphological differences. Differentiated neurons exhibited a s nificant increment in neurite density and neurite length when compared to undifferen ated SH-SY5Y cells ( Figure 1A,B).

GA Treatment Induces Abnormal Tau Phosphorylation and Axon Degeneration in SH-SY5Y-Differentiated Neurons
To develop a neuron-like model of AD bearing Tau hyperphosphorylation, the d ferentiation of SH-SY5Y to neurons was firstly performed as described by Shipley et [26]. SH-SY5Y-derived neurons were subsequently imaged by confocal microscopy (F ure 1A,B) to compare morphological differences. Differentiated neurons exhibited a s nificant increment in neurite density and neurite length when compared to undifferen ated SH-SY5Y cells ( Figure 1A,B).

GA Treatment Induces Abnormal Tau Phosphorylation and Axon Degeneration in SH-SY5Y-Differentiated Neurons
To develop a neuron-like model of AD bearing Tau hyperphosphorylation, the d ferentiation of SH-SY5Y to neurons was firstly performed as described by Shipley et [26]. SH-SY5Y-derived neurons were subsequently imaged by confocal microscopy (F ure 1A,B) to compare morphological differences. Differentiated neurons exhibited a s nificant increment in neurite density and neurite length when compared to undifferen ated SH-SY5Y cells ( Figure 1A,B).

GA Treatment Induces Abnormal Tau Phosphorylation and Axon Degeneration in SH-SY5Y-Differentiated Neurons
To develop a neuron-like model of AD bearing Tau hyperphosphorylation, the differentiation of SH-SY5Y to neurons was firstly performed as described by Shipley et al. [26]. SH-SY5Y-derived neurons were subsequently imaged by confocal microscopy ( Figure 1A following administration of both 0.7 mM GA and 1 mM GA ( Figure 1D). The same trend was recorded on S396 residue, recording a significant increment of phosphorylation levels following 0.7 mM GA and 1 mM GA administration ( Figure 1E). Furthermore, we evaluated whether GA treatment, and resulting Tau hyperphosphorylation, would result in any change in neuronal cell viability. To assess neuronal viability, an MTT assay was performed at 24 h after GA administration, with both 0.7 mM GA and 1 mM GA treatment, resulting in a significant decrease in cell viability ( Figure  1F).  (D) Quantification of phosphorylation ratio pTau/tTau on S199 of SH-SY5Y-differentiated neurons following 0.7 mM GA or 1 mM treatment. ANOVA followed by Tukey's post-hoc test was used to compare differences between different groups. (E) Quantification of phosphorylation ratio pTau/tTau on S396 of SH-SY5Y-differentiated neurons following 0.7 mM GA or 1 mM GA treatment. ANOVA followed by Tukey's post-hoc test was used to compare differences between different groups. (F) Cell viability of SH-SY5Y-differentiated neurons following GA treatment at different concentrations. Ordinary one-way ANOVA followed by Tukey's post-hoc test was used to compare differences between different groups. Data are presented as mean ± SEM. n = 3. * p < 0.05; ** p < 0.01; **** p < 0.0001.
After validating the neuronal differentiation of SH-SY5Y cells, we investigated whether GA treatment for 24 h would result in any AD defects in differentiated neurons, as described by Koriyama in undifferentiated SH-SY5Y cells [33]. Confocal imaging, and subsequent axon length analysis, showed a significant reduction of neurite length in neurons treated with 0.7 mM GA and 1 mM GA when compared to untreated neurons ( Figure 1A,C). In addition to this, an assessment of Tau phosphorylation levels was performed on neurons treated with GA. Tau residue S199 was found to be abnormally phosphorylated following administration of both 0.7 mM GA and 1 mM GA ( Figure 1D). The same trend was recorded on S396 residue, recording a significant increment of phosphorylation levels following 0.7 mM GA and 1 mM GA administration ( Figure 1E).
Furthermore, we evaluated whether GA treatment, and resulting Tau hyperphosphorylation, would result in any change in neuronal cell viability. To assess neuronal viability, an MTT assay was performed at 24 h after GA administration, with both 0.7 mM GA and 1 mM GA treatment, resulting in a significant decrease in cell viability ( Figure 1F).

Novel Compounds Inhibit AChE Enzyme Activity in SH-SY5Y-Differentiated Neurons
Upon validation that GA treatment results in Tau abnormal phosphorylation in SH-SY5Y-derived neurons, we decided to investigate the inhibitory activity of the novel AChE inhibitors that were newly synthesized.
In 0.7 mM GA-exposed neurons we recorded a significant inhibition of AChE activity by compound XJP-1 at a concentration up to 0.5 µM, with the same trend being scored by all other AChE inhibitors investigated ( SH-SY5Y-derived neurons following GA treatment at different concentrations. Ordinary one-way ANOVA followed by Tukey's post-hoc test was used to compare differences between different groups. (D) Quantification of phosphorylation ratio pTau/tTau on S199 of SH-SY5Y-differentiated neurons following 0.7 mM GA or 1 mM treatment. ANOVA followed by Tukey's post-hoc test was used to compare differences between different groups. (E) Quantification of phosphorylation ratio pTau/tTau on S396 of SH-SY5Y-differentiated neurons following 0.7 mM GA or 1 mM GA treatment. ANOVA followed by Tukey's post-hoc test was used to compare differences between different groups. (F) Cell viability of SH-SY5Y-differentiated neurons following GA treatment at different concentrations. Ordinary one-way ANOVA followed by Tukey's post-hoc test was used to compare differences between different groups. Data are presented as mean ± SEM. n = 3. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Novel Compounds Inhibit AChE Enzyme Activity in SH-SY5Y-Differentiated Neurons
Upon validation that GA treatment results in Tau abnormal phosphorylation in SH-SY5Y-derived neurons, we decided to investigate the inhibitory activity of the novel AChE inhibitors that were newly synthesized.
In 0.7 mM GA-exposed neurons we recorded a significant inhibition of AChE activity by compound XJP-1 at a concentration up to 0.5 μM, with the same trend being scored by all other AChE inhibitors investigated (Figure 2A-E). However, Donepezil and compound 24r showed a significant inhibition at 50 nM concentration as well in 0.7 mM GAtreated neurons ( Figure 2B,E).
An investigation of dual AChE/GSK-3 inhibitor 27g showed a significant inhibition at the 5 μM concentration only ( Figure 2F).
A study on 1 mM GA-exposed neurons showed a decrease in AChE activity following treatment with all inhibitors tested at a concentration up to 50 nM, with the exception of SAD-2 ( Figure 3A-F).  (B) Inhibitory activity of compound Donepezil in 0.7 mM GA-exposed neurons. (C) Inhibitory activity of compound SAD-2 in 0.7 mM GA-exposed neurons. (D) Inhibitory activity of compound SAD-6 in 0.7 mM GA-exposed neurons. (E) Inhibitory activity of compound 24r in 0.7 mM GA-exposed neurons. (F) Inhibitory activity of compound 27g in 0.7 mM GA-exposed neurons. Ordinary one-way ANOVA followed by Tukey's post-hoc test was used to compare differences between different groups. Data are presented as mean ± SEM. n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
An investigation of dual AChE/GSK-3 inhibitor 27g showed a significant inhibition at the 5 µM concentration only ( Figure 2F).
A study on 1 mM GA-exposed neurons showed a decrease in AChE activity following treatment with all inhibitors tested at a concentration up to 50 nM, with the exception of SAD-2 ( Figure  (E) Inhibitory activity of compound 24r in 0.7 mM GAexposed neurons. (F) Inhibitory activity of compound 27g in 0.7 mM GA-exposed neurons. Ordinary one-way ANOVA followed by Tukey's post-hoc test was used to compare differences between different groups. Data are presented as mean ± SEM. n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 3.
AChE inhibition by novel compounds at different concentrations in 1 mM GA-exposed SH-SY5Y-derived neurons. (A) Inhibitory activity of compound XJP-1 in 1 mM GA-exposed neurons. (B) Inhibitory activity of compound Donepezil in 1 mM GA-exposed neurons. (C) Inhibitory activity of compound SAD-2 in 1 mM GA-exposed neurons. (D) Inhibitory activity of compound SAD-6 in 1 mM GA-exposed neurons. (E) Inhibitory activity of compound 24r in 1 mM GA-exposed neurons. (F) Inhibitory activity of compound 27g in 1 mM GA-exposed neurons. Ordinary one-way ANOVA followed by Tukey's post-hoc test was used to compare differences between different (B) Inhibitory activity of compound Donepezil in 1 mM GA-exposed neurons. (C) Inhibitory activity of compound SAD-2 in 1 mM GA-exposed neurons. (D) Inhibitory activity of compound SAD-6 in 1 mM GA-exposed neurons. (E) Inhibitory activity of compound 24r in 1 mM GA-exposed neurons.

AChE Inhibition Prevents GA-Induced Tau Phosphorylation on S396 but Not on S199
After validating the inhibitory activity of the novel compounds on the AChE enzyme (Figures 2 and 3), we investigated whether GA-induced Tau hyperphosphorylation could be prevented by AChE inhibition exerted by novel compounds. The exposure of neuronal cells to 0.7 mM GA dramatically increased Tau phosphorylation levels on S199 ( Figure 1D), whilst AChE inhibitor XJP-1, at 5 µM and 0.5 µM concentrations, significantly reduced Tau hyperphosphorylation on S199 in neurons treated with 0.7 mM GA ( Figure 4A), with the same trend being followed by the control AChE inhibitor Donepezil ( Figure 4B). On the other hand, treatment with novel AChE inhibitors SAD-2 did not reduce S199 phosphorylation in 0.7 mM GA-exposed SH-SY5Y-differentiated neurons ( Figure 4C), whilst compounds SAD-6 and 24r recorded a significant reduction of S199 abnormal phosphorylation when administrated at a 5 µM concentration only ( Figure 4D,E). In addition, for SH-SY5Y-differentiated neurons treated with 1 mM GA, none of the AChE inhibitors tested, including Donepezil, showed a significant reduction of Tau phosphorylation on S199 ( Figure S1A-E). phosphorylation when administrated at a 5 μM concentration only ( Figure 4D,E). In addition, for SH-SY5Y-differentiated neurons treated with 1 mM GA, none of the AChE inhibitors tested, including Donepezil, showed a significant reduction of Tau phosphorylation on S199 ( Figure S1A-E).
To further assess the efficacy of the novel compounds in preventing Tau abnormal phosphorylation, another residue, S396, was examined. In neurons treated with 0.7 mM GA, all therapies under study showed a potent effect in reducing S396 phosphorylation, at all concentrations tested ( Figure 5A-E).
An overlapping trend was found in neurons treated with 1 mM GA, with all the novel compounds inducing a significant reduction of phosphorylation levels on Tau S396, at a concentration as low as 10 nM, with the exception of SAD-6 ( Figure 6A-E).  . Tau S199 phosphorylation levels in 0.7 mM GA-exposed neurons after treatment with novel AChE inhibitors. (A) Quantification of phosphorylation ratio pTau/tTau on S199 of 0.7 mM GA-treated SH-SY5Y-differentiated neurons after XJP-1 treatment. (B) Quantification of phosphorylation ratio pTau/tTau on S199 of 0.7 mM GA-treated SH-SY5Y-differentiated neurons after Donepezil treatment. (C) Quantification of phosphorylation ratio pTau/tTau on S199 of 0.7 mM GA-treated SH-SY5Ydifferentiated neurons after SAD-2 treatment. (D) Quantification of phosphorylation ratio pTau/tTau on S199 of 0.7 mM GA-treated SH-SY5Y-differentiated neurons after SAD-6 treatment. (E) Quantification of phosphorylation ratio pTau/tTau on S199 of 0.7 mM GA-treated SH-SY5Y-differentiated neurons after 24r treatment. Kruskal-Wallis test followed by Dunn's post-hoc was used to compare the differences between different groups. Data are expressed as mean ± SEM, n = 3 with at least 5 repetitions per experiment,* p < 0.05; *** p < 0.001, **** p < 0.0001.
To further assess the efficacy of the novel compounds in preventing Tau abnormal phosphorylation, another residue, S396, was examined. In neurons treated with 0.7 mM GA, all therapies under study showed a potent effect in reducing S396 phosphorylation, at all concentrations tested ( Figure 5A-E).  An overlapping trend was found in neurons treated with 1 mM GA, with all the novel compounds inducing a significant reduction of phosphorylation levels on Tau S396, at a concentration as low as 10 nM, with the exception of SAD-6 ( Figure 6A-E).

Dual AChE/GSK-3 Inhibitor 27g Modulates Tau Phosphorylation on Both S199 and S396
To study the effects of treatment with dual inhibitor AChE/GSK-3 27g, Tau phosphorylation on S199, in 0.7 mM GA-treated neurons, was assessed. The novel compound showed a significant reduction of S199 phosphorylation levels when compared to the untreated 0.7 mM GA neurons, in a dose-dependent manner up to 50 nM concentration (Fig-Figure 6. Tau S396 phosphorylation levels in 1 mM GA-exposed neurons after treatment with novel AChE inhibitors. (A) Quantification of phosphorylation ratio pTau/tTau on S396 of 1 mM GA-treated SH-SY5Y-differentiated neurons after XJP-1 treatment. (B) Quantification of phosphorylation ratio pTau/tTau on S396 of 1 mM GA-treated SH-SY5Y-differentiated neurons after Donepezil treatment. (C) Quantification of phosphorylation ratio pTau/tTau on S396 of 1 mM GA-treated SH-SY5Ydifferentiated neurons after SAD-2 treatment. (D) Quantification of phosphorylation ratio pTau/tTau on S396 of 1 mM GA-treated SH-SY5Y-differentiated neurons after SAD-6 treatment. (E) Quantification of phosphorylation ratio pTau/tTau on S396 of 1 mM GA-treated SH-SY5Y-differentiated neurons after 24r treatment. Kruskal-Wallis test followed by Dunn's post-hoc was used to compare the differences between different groups. Data are expressed as mean ± SEM, n = 3 with at least 5 repetitions per experiment, * p < 0.05; *** p < 0.001, **** p < 0.0001.

Dual AChE/GSK-3 Inhibitor 27g Modulates Tau Phosphorylation on Both S199 and S396
To study the effects of treatment with dual inhibitor AChE/GSK-3 27g, Tau phosphorylation on S199, in 0.7 mM GA-treated neurons, was assessed. The novel compound showed a significant reduction of S199 phosphorylation levels when compared to the untreated 0.7 mM GA neurons, in a dose-dependent manner up to 50 nM concentration ( Figure 7A), whilst commercial GSK-3 inhibitor Tideglusib reduced S199 phosphorylation at the 5 µM concentration only ( Figure 7B). However, the novel dual inhibitor was not able to significantly reduce S199 phosphorylation in neurons exposed to 1 mM GA ( Figure S2A), despite the GSK-3 inhibitor Tideglusib successfully reducing S199 phosphorylation at 5 µM and 0.5 µM concentrations ( Figure S2B).
Mol. Sci. 2022, 23, x FOR PEER REVIEW 1 concentrations tested of compound 27g in 1mM GA-exposed neurons, with the sam sults being scored by the control drug Tideglusib ( Figure 9A,B).
To further characterize the efficacy of compound 27g in reducing Tau hyperphosphorylation, residue S396 was examined. A significant decrease in S396 phosphorylation levels was recorded, up to a 50 nM concentration, in 0.7 mM GA-exposed neurons treated with compound 27g, with Tideglusib scoring similar results ( Figure 8A,B). Moreover, inhibition of both AChE and GSK-3 enzymes resulted in a significant reduction for all concentrations tested of compound 27g in 1 mM GA-exposed neurons, with the same results being scored by the control drug Tideglusib (Figure 9A,B).

Dual AChE/GSK-3 Inhibitor 27g Prevents Tau-Induced Neurodegeneration and Protects from Neuronal Cell Death
During AD progression, the formation of aggregated forms of hyperphosphorylated Tau leads to the disruption of neuronal morphology, with jeopardized neural architecture resulting in a loss of brain circuits and functions. Thus, the prevention of neurite degeneration is a fundamental test for a potential AD therapy. We have exploited confocal microscopy to investigate the neuronal morphology in the presence of any ameliorations at 24 h after incubation with 0.7 mM GA and compound 27g at various concentrations. We found that compound 27g significantly prevented neurite shortening in a dose-dependent manner and exhibited more potent effects than Tideglusib at the same concentrations ( Figure 10A,B).
The inhibition of both AChE and GSK-3 by compound 27g confirmed the positive trend by preventing neurite shortening in 1 mM GA-treated neurons as well, overlapping Tideglusib results ( Figure 11A,B). In addition to this, we observed an improvement of cell viability at both concentrations of GA following administration of 27g or Tideglusib ( Figure S3A-D). Conversely, treatment with AChE inhibitors resulted in variable results in preventing neurodegeneration, both in terms of morphology protection (Figures S4 and S5) and cell viability (Figures S6 and S7). Figure 8. Reduction of Tau S396 phosphorylation after 27g treatment in 0.7 mM GA-exposed neurons. (A) Quantification of phosphorylation ratio pTau/tTau on S396 of 0.7 mM GA-treated SH-SY5Y-differentiated neurons after 27g treatment. (B) Quantification of phosphorylation ratio pTau/tTau on S396 of 0.7 mM GA-treated SH-SY5Y-differentiated neurons after Tideglusib treatment. Kruskal-Wallis test followed by Dunn's post-hoc was used to compare the differences between different groups. Data are expressed as mean ± SEM, n = 3 with at least 5 repetitions per experiment; ** p < 0.01, **** p < 0.0001. Figure 9. Reduction of Tau S396 phosphorylation after 27g treatment in 1 mM GA-exposed neurons. (A) Quantification of phosphorylation ratio pTau/tTau on S396 of 1 mM GA-treated SH-SY5Y-differentiated neurons after 27g treatment. (B) Quantification of phosphorylation ratio pTau/tTau on S396 of 1 mM GA-treated SH-SY5Y-differentiated neurons after Tideglusib treatment. Kruskal-Wallis test followed by Dunn's post-hoc was used to compare the differences between different groups. Data are expressed as mean ± SEM, n = 3 with at least 5 repetitions per experiment, *** p < 0.001, **** p < 0.0001.

Dual AChE/GSK-3 Inhibitor 27g Prevents Tau-Induced Neurodegeneration and Protects from Neuronal Cell Death
During AD progression, the formation of aggregated forms of hyperphosphorylated Tau leads to the disruption of neuronal morphology, with jeopardized neural architecture resulting in a loss of brain circuits and functions. Thus, the prevention of neurite degeneration is a fundamental test for a potential AD therapy. We have exploited confocal microscopy to investigate the neuronal morphology in the presence of any ameliorations at 24 h after incubation with 0.7 mM GA and compound 27g at various concentrations. We found that compound 27g significantly prevented neurite shortening in a dose-dependent manner and exhibited more potent effects than Tideglusib at the same concentrations (Figure 10A,B).
The inhibition of both AChE and GSK-3 by compound 27g confirmed the positive trend by preventing neurite shortening in 1 mM GA-treated neurons as well, overlapping Tideglusib results ( Figure 11A,B). In addition to this, we observed an improvement of cell viability at both concentrations of GA following administration of 27g or Tideglusib (Figure S3A-D). Conversely, treatment with AChE inhibitors resulted in variable results in preventing neurodegeneration, both in terms of morphology protection (Figures S4 and  S5) and cell viability ( Figures S6 and S7).  Ordinary one-way ANOVA followed by Tukey's post-hoc test was used to compare differences between different groups. Data are presented as mean ± SEM, n = 3 with at least 5 repetitions per experiment, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Figure 10. Dose-dependent amelioration of neurite morphology by compound 27g in 0.7 mM GAexposed neurons. (A) Neurite length quantification of 0.7 mM GA-treated SH-SY5Y-differentiated neurons after 27g treatment. Scale bar 100 μM. (B) Neurite length quantification of 0.7 mM GAtreated SH-SY5Y-differentiated neurons after Tideglusib treatment. Ordinary one-way ANOVA followed by Tukey's post-hoc test was used to compare differences between different groups. Data are presented as mean ± SEM, n = 3 with at least 5 repetitions per experiment, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Discussion
In AD, the regulation of multiple pathways involved in the pathogenesis and progression of the disease is the main target for a potential disease-modifying therapy. The novel compound 27g aims to do so by inhibiting both enzymes of AChE and GSK-3 simultaneously, with its structure designed using Tacrine and Pyrimidone as moieties [37]. Ordinary one-way ANOVA followed by Tukey's post-hoc test was used to compare differences between different groups. Data are presented as mean ± SEM, n = 3 with at least 5 repetitions per experiment,* p < 0.05; ** p < 0.01; **** p < 0.0001.

Discussion
In AD, the regulation of multiple pathways involved in the pathogenesis and progression of the disease is the main target for a potential disease-modifying therapy. The novel compound 27g aims to do so by inhibiting both enzymes of AChE and GSK-3 simultaneously, with its structure designed using Tacrine and Pyrimidone as moieties [37]. The exploitation of a cell model that well recapitulates AD neuronal features is an important approach to better characterize newly synthesized compounds for AD treatment. We have successfully combined two different protocols, developed and validated a neuronal cell model of AD bearing Tau hyperphosphorylation (Shipley 2016;Koriyama et al., 2015). In particular, using GA to inhibit glycolysis, and increase AGEs level, well recapitulates the AD human features, in which the accumulation of AGEs and subsequent activation of the RAGE receptor is often described, leading to the activation of the amyloidogenic pathway and Tau hyperphosphorylation [1,[38][39][40][41]. The accumulation of AGEs as a consequence of GA treatment results in Tau disease, as seen in diabetes, of which the pathology is often linked to the development of AD. The model developed recapitulates the neuronal features of AD well, with GA treatment leading to abnormal phosphorylation of Tau protein on different residues and neurite degeneration, along with reduced cell viability. This model is of peculiar relevance as it offers a novel platform to further study RAGE signalling, which has often been linked to AD development [8]. RAGE is an immunoglobulin superfamily receptor capable of binding AGEs, Amyloid beta, and other ligands. In a study involving both SH-SY5Y and hippocampal primary neurons, the application of AGEs induced a dose-dependent increase on Tau hyperphosphorylation on multiple residues, including S199, S396, S404 [42]. These results overlap our findings obtained after GA treatment, showing an increase in aberrant phosphorylation in Tau S199 and S396. The AChE inhibitory compounds (XJP-1, SAD-2, and SAD-6) with a 1-benzyl-pyridin-1-ium bromide backbone structure and with the pharmacophore skeleton 1-benzyl-pyridin-1-ium (Table 1) were evaluated with this cell model. Among these compounds, the in silico predicted potency order is SAD-2 > XJP-1 > SAD-6, suggesting the influence of these substituents is Se > O > S, which did not reflect a similar result upon completion of biological assays. In addition, another sulfone analogue of Donepezil, compound 24r, was screened by this cell model, and the results showed that the 24r structure backbone is best compared to XJP or SAD, but again failed to produce any further beneficial effects when compared to the other AChE inhibitors. The dual AChE/GSK-3 inhibitor, compound 27g, with a Tacrine-Pyrimidone skeleton, was active in the cell model, and also displayed potent in vivo anti-AD efficacy. The assessment of dual AChE/GSK-3 inhibitor 27g showed promising results, with overlapping results obtained by the commercially available GSK-3 inhibitor Tideglusib. Therefore, the simultaneous inhibition of AChE and GSK-3 improves the efficacy in vivo [37]. On the other hand, assessment of different single-targeting AChE inhibitors did not result in a homogenous comparable outcome, despite significant inhibition of the enzyme recorded in vitro [43], suggesting that inhibition of the AChE enzyme may modulate different AD pathways involved in the Tau hyperphosphorylation triggered by GA, but in a non-specific manner derived by the distribution and concentration of different acetylcholine receptors. Finally, for several compounds, a modest inhibition of AChE enzyme still resulted in a significant amelioration of the parameters analyzed, further suggesting that inhibition of the AChE enzyme results in a response controlled by the pathways activated by specific acetylcholine receptors.

Reagents
AChE inhibitor Donepezil and GSK-3β inhibitor tideglusib were purchased from Sigma-Aldrich Ltd (St. Louis, MO, USA). Drug candidates of novel AChE inhibitors XJP-1, SAD-2, SAD-6, and 24r and 27g were gifted by Prof. Jinyi Xu (China Pharmaceutical University, Nanjing, China). All drug compounds were dissolved in DMSO to make a stock solution with a final concentration of 1 mM and stored at −20 • C. The compound structure is outlined in Table 1.

SH-SY5Y Culture and Neuronal Differentiation
SH-SY5Y cells were cultured on different types of media, described in Table 2, and differentiated as described by Shipley with some modifications [26]. Briefly, SH-SY5Y cells were plated with Basic Growth media and allowed to reach 70% confluency. Subsequently, the medium was changed to Differentiation Media-1 and replaced every 48 h for the following 7 days. Cells were then split 1:1 and moved into fresh flasks/dishes and Differentiation Media-2 was added. Differentiation Media-2 was then replaced every 48 h for the following 4 days. Subsequently, the medium was changed for Differentiation Media-3 and replaced every 48 h for the following 7 days. After this period, SH-SY5Y-derived neurons were used for the subsequent assays and analysis.

Glyceraldehyde Induced Tau Hyperphosphorylation
In order to induce Tau hyperphosphorylation, differentiated SH-SY5Y were treated with either 0.7 mM or 1 mM GA for 24 h as previously described [33]. After treatment with GA for 24 h, cells were used for downstream analysis.

Immunostaining Cell Culture
SH-SY5Y cells and differentiated SH-SY5Y cells were fixed in 4% PFA for 10 min at room temperature. Each sample was washed three times with 0.1% PBS-T for 2 min. Following fixation, cells were incubated at room temperature for 2 h in 5% NGS-T blocking solution and incubated overnight at +4 • C with the mouse monoclonal anti-tubulin III primary antibody (#ab179513, Abcam plc., Camabridge, UK) at a final concentration of 1:1000. The primary antibody was then removed, and each sample was washed three times with 0.1% PBS-T. Rabbit anti-mouse secondary antibody Alexa Fluor 488 (#ab150113, Abcam plc., Cambridge, UK) was added at a final 1:2000 concentration and left to incubate at RT for 1 h. The secondary antibody was removed, and each sample was washed three times with 0.1% PBS-T. Samples were then mounted on to glass slides, using FluoroGel Mounting media (Genetex, Irvine, CA, USA). Samples were finally imaged by confocal microscopy within 24 h.

Confocal Microscopy
Cell images were acquired using Zeiss880 Confocal Microscope (Zeiss, Jena, Germany). Laser power was set at 4%, gain 650, digital offset at 350. Acquisition speed was set at 1.3 m, averaging two times per image. Z-stack images at distance of 5 µm were acquired. Analysis of confocal images was performed using ImageJ v2 plug-in NeuronJ v1. Three more random areas were analyzed. A threshold mask was applied to visualize the axon only and not the cell body. Subsequently, the measurement of the axon length was taken, and the distance between coordinates was measured with NeuronJ.

pTAU Quantification
Phosphorylation levels of Tau protein on Serine 199 (S199) and Serine 396 (S396) were quantified using Enzyme Linked Immunosorbent Assay (ELISA) methodology. Following the manufacturer recommended protocol, ELISA kits KHB7041, KHB7031, and KHB0041 (ThermoFisher Scientific, Waltham, MA, USA) were used to quantify phosphorylated Tau S199, phosphorylated S396, and total Tau, respectively. Phosphorylation percentage was obtained for the analyzed residues by normalization against total Tau.

AChE Activity Assay (Ellman's Method)
AChE enzymatic activity was assessed using AChE assay kit (#ab138871, Abcam plc.) following the manufacturer's instructions. A total of 10 5 per well were seeded into a clear 96-well plate. After appropriate differentiation and treatment, cell culture medium was removed, and 100 µL of lysis buffer was added into each well and left to incubate for 15 min at room temperature (RT). Subsequently, 50 µL of acetylthiocholine reaction mixture (1× assay buffer, 1× DTNB stock solution, 1× acetylthiocholine stock solution) was added to each well and samples were left to incubate for 30 min at RT. Samples were then analyzed using a 96-well microplate reader at OD= 410 ± 5 nm. In order to avoid false positives given by butyrylcholinesterase activity, the specific AChE inhibitor Donepezil hydrochloride was used as control.

Cell Viability Assay
A total of 10 5 per well were seeded into a 96-well plate. After SH-SY5Y differentiation, cells were treated with either 0.7 mM or 1 mM GA, respectively. Along with GA treatment, an appropriate concentration of the compound to be tested was added. After 24 h, 25 µL of 5 mg/mL Methylthiazolyldiphenyl-tetrazolium bromide (MTT) (#M2128-100MG, Sigma-Aldrich, St. Louis, MO, USA) was added into each well without removing cell culture media and incubated at 37 • C with 5% CO 2 for 2 h. Subsequently, 100 µL of lysing buffer (50% SDS solution, 25% DMF, 25% demineralized water) was added. After an overnight incubation (20 h) at 37 • C, the OD at 490 nm was measured using 96-well plate reader. The medium/MTT/lysing buffer incubated under the same conditions was used as the control.

Data Analysis
Statistical analysis was performed using GraphPad Prism 9 software. Data obtained were firstly tested for normality using the Shapiro-Wilk test. The Kruskal-Wallis test followed by Dunn's post-hoc was used to compare the differences between three or more groups in non-normally distributed data. The ANOVA test was used to compare the differences between three or more groups of normally distributed samples. Each experiment was performed in triplicate and all results are presented as mean ± standard error of the mean or mean ± standard deviation. Results with a p-value < 0.05 were considered as statistically significant.

Conclusions
In the present work, we provided an upgraded version of the Tau hyperphosphorylation model initially designed by Koryiama [33] on undifferentiated SH-SY5Y. We screened different AChE inhibitors along with the already reported 24r and 27g compounds [37,43], and for both we expanded the investigation on 1 mM GA-treated cells. The screening has confirmed the variability of AChE inhibitors on modulating AD features, whilst suggesting that a dual GSK-3/AChE inhibition strategy could be a potential route for the development of an AD therapy with potential disease-modifying properties. Our finding provides the basis for further studies in more complex AD models, i.e., AD risk gene-edited neurons or astrocytes, to investigate different pathological features such as amyloid pathology and synapsis activation.